ATOMIC SPECTROSCOPY

Strengths:
a. mass spectrum - a distribution of  Flame photometry provides a robust,
ions shown by the use of a mass spectrograph cheap and selective method based in
or mass spectrometer relatively simple instrumentation for
b. alkali metals - any of the elements quantitative analysis of some metals.
lithium, sodium, potassium, rubidium, cesium,
and francium, occupying Group IA (1) of the Limitation:
periodic table  Only applicable to the determination of
c. alkaline earth metals - any of the alkali and some alkaline earth metals.
elements beryllium, magnesium, calcium,
strontium, barium, and radium, occupying Introduction
Group IIA (2) of the periodic table  plays an important role in the control
of sodium, potassium, barium, calcium
and lithium
ATOMIC SPECTROSCOPY  Atoms contain various energy states
 is the determination of elemental and the normal unexcited state is the
composition by its electromagnetic or mass ground state. If energy is gained by the
spectrum atom, this electron may be excited to a
higher state and then subsequently
Three techniques: lose its excess energy by falling back to
(1) atomic emission spectrophotometry (AES), a lower energy orbital.
(2) atomic absorption spectrophotometry (AAS)
(3) atomic fluorescence spectrophotometry Instrumentation
(AFS)

1. Flame – the metal is volatilized in a natural

How the three techniques are implemented. gas/compressed air flame at 2000ºC
- a higher temp.
Atomic Emission Spectrophotometry (2500ºC) may be obtained using air/acetylene
and is required for analysis of Mg by AES.
Principles: 2. Monochromator/Filter – where radiation
 Atoms are thermally excited so that emitted by the excited atoms is passed
they emit light and the radiation 3. Detector – measures the intensity of the
emitted is measured. selected radiation using a photosensitive cell

Applications:
 Quantification of alkali metals in: alkali Interferences
metal salts, infusion and dialysis 1. Ionisation
solutions.  an equilibrium and may be shifted
 Determination of metallic impurities in to the left by addition of another readily ionized
some of the inorganic salts used in element to the sample (produces electrons)
preparing these solutions. Ex: strontium chloride is added in the assay of
effervescent KCl tablets specific method of analysis useful in
some aspects of quality control.
2. Viscosity
 can either increase or decrease the Limitations:
rate of emission  Only applicable to metallic elements.
Ex: sucrose decreases the rate, ethanol  Each element requires a different
increases the rate hollow cathode lamp for its
determination.
3. Anionic interference
 reduces the reading of the sample Introduction:
 may be removed by the addition of  The energy difference between their
lanthanum chloride ground state orbital and the excited
state is too great for thermal
excitation. If the energy is too great,
Atomic Absorption Spectrophotometry AAS may be used.

Principles: Instrumentation
 Atoms of a metal are volatilized in a
flame and their absorption of a narrow
band of radiation produced by a hollow
cathode lamp, coated with the
particular metal being determined, is
measured.
 All atoms can absorb light.
 The wavelength at which light is
absorbed is specific for each element. Parts of Atomic Absorption
If a sample containing nickel, for
Spectrophotometry.
example, together with elements such
as lead and copper is exposed to light 1. Light source – hollow cathode lamp
at the characteristic wavelength for 2. Flame – air/acetylene (2500ºC); nitrous
nickel, then only the nickel atoms will oxide/acetylene (3000ºC) for Al or Ca
absorb this light. 3. Monochromator – narrows down the width
 The amount of light absorbed at this of the band of radiation being examined
wavelength will increase as the number 4. Detector – a photosensitive cell
of atoms of the selected element in the
light path increases, and is proportional
to the concentration of absorbing Examples
atoms. 1. Assay of Calcium and Magnesium in
 The relationship between the amount Haemodialysis Fluid
of light absorbed and the 2. Assay of Lead in Sugars
concentration of the analyte present in 3. Assay of Mg and Sr in Calcium acetate
known standards can be used to 4. Assay of Palladium in Carbenicillin Sodium
determine unknown concentrations by 5. Assay of Copper and Iron in Ascorbic acid
measuring the amount of light they
absorb. An atomic absorption
spectrometer is simply an instrument
in which these basic principles are
applied to practical quantitative
analysis.

Applications:
 Determination of metal residues
remaining from the manufacturing
process in drugs.
Strengths:
 More sensitive than AES. A highly
CHROMATOGRAPHY supporting medium that interacts with the
analytes
Some materials appear homogenous, but are c. supporting medium: a solid surface on which
actually a combination of substances. For the stationary phase is bound or coated
example, green plants contain a mixture of
different pigments. In addition, the black ink in
the pens that are used in this experiment is a
mixture of different colored materials. In many
instances, we can separate these materials by Categories of Chromatography and Their
dissolving them in an appropriate liquid and Relationship to Each Other.
allowing them to move through an absorbent
matrix, like paper. Chromatography is a method
used by scientists for separating organic and
inorganic compounds so that they can be
analyzed and studied. By analyzing a compound,
a scientist can figure out what makes up that
compound. Chromatography is a great physical
method for observing mixtures and solvents.
The word chromatography means "color
writing" which is a way that a chemist can test
liquid mixtures. While studying the coloring
materials in plant life, a Russian botanist
invented chromatography in 1903. His name
was M.S. Tswett.

a. chromatogram - a visible record Types:

(such as a series of colored bands, or a graph) Chromatography can be classified based on the
showing the result of separation of the type of mobile phase, stationary phase and
components of a mixture by chromatography support material:
b. retention factor (Rf) – vale that can 1.) The primary division of chromatographic
quantify the amount that each component of a techniques is based on the type of mobile phase
mixture travel used in the system:
c. chromophore - an atom or group
whose presence is responsible for the color of a Type of Chromatography
compound Gas chromatography (GC)
Liquid chromatograph (LC)

CHROMATOGRAPHY Type of Mobile Phase

 a separation technique based on the gas
different interactions of compounds with two liquid
phases, a mobile phase and a stationary phase,
as the compounds travel through a supporting
medium 2. ) Further divisions can be made based on
the type of stationary phase used in the system:

Gas Chromatography

Name of GC Method Type of Stationary

Phase

Gas-solid solid, underivatized

chromatography support
Components:
a. mobile phase: a solvent that flows through
the supporting medium Gas-liquid liquid-coated
b. stationary phase: a layer or coating on the chromatography support
 Bonded-phase gas chemically- (b) a sufficiently narrow width of the solute
chromatography derivatized support peaks (i.e, good efficiency for the separation
system)

2.) Solute Retention:

A solute’s retention time or retention
Liquid Chromatography volume in chromatography is directly related to
the strength of the solute’s interaction with the
Name of LC Method Type of Stationary mobile and stationary phases.
Phase Retention on a given column pertain to
the particulars of that system:
- size of the column
Adsorption solid, underivatized - flow rate of the mobile
chromatography support phase
Capacity factor (k’): more universal
Partition liquid-coated or
measure of retention, determined from tR or VR.
chromatography derivatized support
It is useful for comparing results obtained on
Ion-exchange support containing different systems since it is independent on
chromatography fixed charges column length and flow-rate.

Size exclusion porous support 3.) Efficiency:

chromatography Efficiency is related experimentally to a
solute’s peak width.
Affinity support with - an efficient system will
chromatography immobilized ligand produce narrow peaks
- narrow peaks  smaller
3.) Chromatographic techniques may also be
difference in interactions in order to separate
classified based on the type of support material
two solutes
used in the system:
Efficiency is related theoretically to the
 Packed bed (column) chromatography
various kinetic processes that are involved in
 Open tubular (capillary) chromatography
solute retention and transport in the column
 Open bed (planar) chromatography
- determine the width or
standard deviation (s) of peaks

4.) Measures of Solute Separation:

- separation factor (a) – parameter
used to describe how well two solutes are
separated by a chromatographic system
- resolution (RS) – resolution between
two peaks is a second measure of how well two
peaks are separated

Different types of support system used in

chromatography. THIN-LAYER CHROMATOGRAPHY
• This is used to determine impurities in
Theory of Chromatography:
pharmaceutical raw materials and
1.) Typical response obtained by
formulated products.
chromatography (i.e., a chromatogram):
• Has been developed as a fundamental
chromatogram -
quality control technique for trace of
concentration versus elution time
impurities.
• The sophistication in the application of
The separation of solutes in chromatography
the technique derives from the broad
depends on two factors:
choice of stationary phase, mobile
(a) a difference in the retention of solutes (i.e., a phase and the wide range of spray
difference in their time or volume of elution reagents which can use of high-
 pressure TLC and interfacing with travels up the plate in a given time.
detection systems such as RAMAN
SPECTROSCOPY and MASS
SPECTROMETRY. FORMULA:
• Often used as a basic identity check on Rf = distance traveled by
pharmaceutical raw materials. sample/distance traveled by solvent

Stationary phases
PRINCIPLE • Silica gel is the most commonly used
• An analyte migrates up or across a adsorbant for TLC. The rate at which
layer of stationary phase (most compounds migrate up a silica gel plate
commonly silica gel), under the depends on their polarity. In a given
influence of a mobile phase (usually a length of time, the most polar
mixture of organic solvent) which compounds move at least distance up
moves through the stationary phase by the plate while the least polar move
capillary action. The distance moved by the furthest.
the analyte is determined by its • Other used polar stationary phase
relative affinity for the stationary vs the - Silica Gel G
mobile phase. - Silica Gel GF254
- Cellulose
Instrumentation - Keiselguhr G

DETECTION OF COMPOUNDS ON TLC PLATES

FOLLOWING DEVELOPMENT
1. ULTRAVIOLET LIGHT
- Light with wavelength of 254 nm is
used to illuminate the plate and if the analyte
absorbs UV light it can be seen as black spot on
a yellow background where it quenches the
fluorescence of the background. For example,
the pharmacopoeial test for anthraquinones in
Set-up for Thin Layer Chromatography. aloes observes the fluoroscenes of these
compounds under UV light at 365 mm.
The most frequently used system is a glass or
2. IODINE VAPOUR
plastic plate coated with silica gel; from the
- The plate is put into a tank containing
application the silica gel particle size is in the
iodine crystals. This treatment will produce
range 2-25 um.
brown spots with many organic compounds;
Iodine spots may be sprayed with starch
The method of use for this system is as follows:
solution in order to stain them permanently.
i. A few ul of sample solution are slowly
Iodine is used as a location agent in
spotted onto the plate at the origin. If
pharmacopeial TLC test of fixed oils and of
more than ca 1 ul is applied at once,
cetrimide.
the spot will spread too far. The spot
3. POTASSIUM PERMANGANATE
has to be allowed to dry between each
- Provides a method for the detection
application of 1 ul. Loadings of sample
of sugars and sugar-like molecules. It used in
are typically 20 ug.
TLC identity checks for the antibacterial agents
ii. The bottom 0.5 cm of the plate is
clindamycin and lincomycin and in a check for
immersed in the mobile phase
related substances and streptomycin.
contained in a tank and the liquid
mobile phase is allowed to travel up
4. NINHYDRIN SOLUTION
the silica gel plate by capillary action.
- this reagent gives pink spots with
iii. The more polar a compound is the
primary amines and yellow spots with tertiary
more it adsorbs the silica gel stationary
amines. This is used in pharmacopeial identity
phase, the less time it spends in the
test for some aminoglycoside antibiotics such as
mobile phase it travels up the plate
gentamycin, in a limit test for aminobutanol in
and thus the shorter the distance it
ethambutol and can be used as a general screen introduction endures quantitative precision.
for nitrogen containing drugs in conjuction with 2. HPLC is the chromatographic technique which
Dragendorff reagent. Dragendorff reagent will has seen the most intensive development in
produce orange spots with tertiary amines and recent years, leading to improved columns,
may be used to overspray plates which have detectors and software control.
been sprayed in the first instance with 3. The variety of columns and detectors means
ninhydrin. that the selectivity of the method can be readily
adjusted.
5. ALKALINE TETRAZOLIUM BLUE 4. Compared to GC, there is a less risk of sample
- Quite specific for corticosteroids, degradation because heating is not required in
producing blue spots on a white background. the chromatographic process.
The tetrazolium spray is used in a test for 5. It is readily automated.
related foreign steroids in fluclorolone 6. With the advent of UPLC methods can be very
acetonide. rapid.

6. ETHANOLSULPHURIC ACID 20% Limitations

- Is used to produce fluorescent spots 1. There is still a requirement for reliable and
from corticosteroids such as dexamethasone or inexpensive detectors which can monitor
prednisolone by spraying the plate, heating to compounds that lack a chromophore.
120 degrees Celsius and then observing the 2. Drugs have to be extracted from their
plate under UV light at 365 nm. formulation prior to analysis.
3. Large amount of organic solvent waste are
generated, which are expensive to dispose of.

HIGH-PERFORMANCE LIQUID
Instrumentation
CHROMATOGRAPHY

Principles
A liquid mobile phase is pumped under
pressure through a stainless steel column
containing particles of stationary phase with a
diameter of 3-10 mcm (1.7 mcm in UPLC). The
analyte is loaded onto the head of the column
via a loop valve and separation of a mixture
occurs according to the relative lengths of time
spent by its components in the SP. It should be
noted that all components in a mixture spend
more or less the same time in the MP in order
to exit the column.

Applications
1. The combination of HPLC with monitoring by
UV/Vis detection provides an accurate, precise
and robust method for quantitative analysis of
pharmaceutical products and is the industry
standard method for this purpose.
2. Monitoring of the stability of pure drug
substances and of drugs in formulations, with
quantitation of any degradation products.
3. Measurement of drugs and their metabolites
in biological fluids.
4. Determination of partition coefficient and
pKa values of drugs and of drug protein binding.

Strengths
1. Easily controlled and precise sample
 produce retardation of a compound passing
through a column.
1. Straight Phase (silica gel) – by
adsorption
2. Reversed Phase (ODS silica gel) – by
partitioning according to lipophilicity

The more polar a mobile phase, the more

quickly it will elute a compound from a silica gel
column, and the more lipophilic a mobile phase,
the more quickly it will elute a compound from
reversed-phase column

Structural factors which govern rate of elution

of compounds from HPLC columns
Elution of Neutral Compounds
◦ For a neutral compound it is he balance
between its polarity and lipophilicity
which will determine the time it takes
for it to elute from an HPLC column;
the pH of the mobile phase does not
play a part.
◦ In the case of a reversed-phase
column, the more lipophilic a
compound is the more it will be
retained. For a polar column such as a
silica gel column, the more polar a
compound is the more it will retained.
◦ When these compounds are eluted
from a reverse-phase column using a
mobile phase containing
methanol/water, the expected order of
elution would be:
Prednisolone, Betamethasone,
Parts of a HPLC machine. Betamethasone 17-valerate,
Betametahsone 21-valerate and
Betamethasone diproprionate
1. a solvent reservoir
2. a pump (pressure of 4000 psi and at flows of
up to 10 ml/min) Basic comparison between HPLC and GC.
3. a loop injector (20 mcl is the usual standard
or 1-200 mcl)
4. a column (usually packed with
octadecylsilane-coated silica gel)
5. a detector (UV/Vis)
6. a data capture system (computing integrator
or a PC with suitable software)
7. tubing connections (column to the injector
and detector with internal diameter ca 0.2mm)
8. more advanced instruments:
a. automatic sample injection
b. automatic column oven

Stationary and mobile phases

There are two principal mechanism which
 for the analysis of sugars and surfactants.

6. Cyanopropyl silica gel

– A moderately polar phase applicable to
the analysis of surfactants.

7. Strong cation exchanger (SCX)

– usually based on ion pairing of the
analyte with sulfonic acid groups on the surface
of the stationary phase. Useful for analysis of
very polar compounds such as aminoglycosides
and other charged sugar molecules and polar
bases such as catecholamines.

8. Strong anion exchanger (SAX)

– usually based on ion pairing of the
analyte with quaternary ammonium groups on
the surface of the stationary phase. Useful for
the separation of polar compounds with anionic
Some commonly used HPLC stationary phase groups such as nucleotides and anionic drug
metabolites such as sulphates or glucuronides.
1. Octadecylsilyl (ODS) silica gel
Some detectors commonly used in HPLC
– the most commonly used phase,
◦ Variable wavelength UV detector
applicable to most problems in analysis of
◦ Diode array detector (DAD)
pharmaceutical formulations. It can be also
◦ Evaporation light-scattering detector
applied to the analysis of peptides, where wide-
(ELSD)
pore packings are used to improve access these
◦ Electrochemical detector
bulky molecules to the internal surface of the
◦ Pulsed amperometric detector
packings.
◦ Refractive index detector (RI)
◦ Flourescence detector
2. Octyl silane and butyl silane silica gel
Corona charged aerosol detector (CAD)
– useful alternatives to ODS phases. The
shorter hydrocarbon chains do not tend to lead GAS CHROMATOGRAPHY
to shorter retentions times of analysis since the
carbon loading on and retention time is also Principles
dependent on how much of the stationary A gaseous mobile phase flows under
phase is accessible to partitioning by the pressure through a heated tube either coated
analyte. with a liquid SP or packed with liquid SP coated
onto a solid support. The analyte is loaded onto
3. Phenyl silane silica gel the bead of the column via a heated injection
– useful for slightly more selective analysis port, where it evaporates. It then condenses at
of compounds containing large numbers of the head of the column, which is at lower
aromatic rings, e.g. propranolol phenyl groups temperature. The oven temperature is then
on the stationary phase if the analyte is either held constant or programmed to rise
deficient electrons e.g. nitro compounds. gradually. Once on the column, separation of a
mixture occurs according to the relative lengths
4. Silica gel of time spent by its components in the SP.
– often used in the past for problematical
compounds but, with gradual improvement of
reverse phases, increasingly less compounds
such as in the separation of different classes of Applications
lipids and in the analysis of surfactant, which • The characterization of some
tend to form micelles under the conditions used unformulated drugs, particularly with
for reverse-phase chromatography. regard to detection of process
impurities.
5. Aminopropyl silica gel • Limit tests for solvent residues and
– A moderately polar phase often used other volatile impurities in drug
 substances. 1. injection of the sample – manually or by
• Sometimes used for quantification of autosampler
drugs in formulations, particularly if the - through a re-
drug lacks a chromophore. sealable septum
• Characterisation of some raw materials 2. The sample is evaporated in the heated
used in synthesis of drug molecules. injection port area and condenses on the head
• Characterisation of volatile oils (which of the column.
may be used as excipients in 3. column – either capillary or packed (mobile
formulations), proprietary cough phase – N or He)
mixtures and tonics, and fatty acids in 4. oven – ambient and ca 400ºC)
fixed oils. 5. detector – flame ionization detector
Measurement of drugs and their metabolites in SYRINGES
biological fluids. • volumes injected is 0.5-2 mcL
• usual volume are 5 mcL and 10 mcL
Limitations • Fill the syringe with about 0.5 mcL of
• Only thermally stable and volatile solvent and draw this solvent into the
compounds can be analysed. barrel slightly before filling with
• The sample may require derivatization sample.
to convert it to a volatile form, thus • Draw the sample to the barrel.
introducing and extra step in analysis • Left the syringe into the injector for a
and, potentially, interferants. couple of seconds to warm up before
• Quantitative sample introduction is the plunger is depressed.
more difficult because of the small • Withdraw the syringe immediately
volumes of sample injected. from the injection port.
• Aqueous solutions and salts cannot be
injected into the instruments.
Injection Systems
Advantages 1. Packed column injections – injection port is
• Requires only very small samples with held at 150-250º
little preparation - direct injection of
• Good at separating complex mixtures 0.1-10 mcL sample is made onto the head of the
into components column
• Results are rapidly obtained (1 to 100 - presents less of a
minutes) problem since all the sample is introduced into
• Very high precision the packed column
• Only instrument with the sensitivity to
detect volatile organic mixtures of low 2. Splits/Splitless Injection – used in conjuction
concentrations with capillary column
• Equipment is not very complex - injection takes place into a
(sophisticated oven) heated glass or quartz liner

3. Cool-on column injection – requires a syringe

Instrumentation with a very fine fused-silica needle
Parts of Gas Chromatography. Advantages:
a. reduced discrimination between
components in mixtures
b. no sample degradation in a hot injector
c. no backflash
Disadvantages:
a. samples have to be clean otherwise
residues will be deposited on the
column
b. the injector is mechanically more
complex and requires more
maintenance than a septum injection
system
c. the syringe needle may damage the
 head of the column

4. Programmable temperature vapouriser –

designed to enable the injection of large
volumes of sample onto capillary GC columns
- sample volume is 5-50 mcL
- liner has diameter of 1mm and is
made of Silicosteel

GC oven
 with fan (ensures uniform heat distribution)
 ranges from 1ºC/min to 4ºC/min
GC Detectors
Types of column 1. Flame ionization
1. Packed columns 2. Electron capture
- usually made from glass which is 3. Nitrogen phosphorus
silanized to remove polar silanol from 4. Thermal conductivity
its surface 5. Radiochemical
- have internal diameters of 2-5 mm 6. Microwave-induced plasma atomic
- packed with particles of a solid support emission
which are coated with the liquid SP 7. Fourier transform IR
 diatomaceous earth (Ca silicate)
- MP is nitrogen with a flow rate of ca 20
ml/min
- produces a relatively low degree of
resolution
- high temperature limit is ca 280ºC
2. Capillary columns
- made from fused silica (coated on the outside
with polyamide for column flexibility)
- internal diameter is 0.15-0.5 mm
- wall is coated with the liquid SP
(thickness is 0.1-5 mcm)
- most common type of coating is based
on organo-silicone polymers
- carrier gas is He (with flow rate
between 0.502ml/min

Summary of parameters governing capillary GC

performance
1. Column temperature
2. Column length
3. Film thickness phase loading
4. Internal diameter